Targeted Testing

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

Glutaric acidemia II patients are split between those with causative variants in the ETFA, ETFB and ETFDH genes, and it is difficult to estimate the fraction with variants in each gene. However, to date all reported causative variants in the ETFB gene are detectable via direct sequencing, and the majority of ETFB patients reported in the literature have been found to have two causative ETFB variants. For example, collective date from four studies shows that approximately 16 causative ETFB variants were found in a total of 18 studied ETFB alleles (Curcoy et al. 2003; Olsen et al. 2003; Schiff et al. 2006; Yotsumoto et al. 2008).

Deletion/Duplication Testing via aCGH

Pricing Comment

# of Genes Ordered

Total Price

1

$690

2

$730

3

$770

4-10

$840

11-30

$1,290

31-100

$1,670

Over 100

Call for quote

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

To date, no large deletions or duplications have been described in the ETFB gene, although most studies of glutaric acidemia type II patients that included ETFB analysis have not been reported to have included deletion and duplication testing.

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Clinical Features

Glutaric acidemia (GA) type II, also known as multiple acyl-CoA dehydrogenase deficiency (MADD), is an inherited disorder of fatty acid and amino acid oxidation. GA type II is caused by defects in one of three genes (ETFA, ETFB or ETFDH). Clinical and biochemical features are not typically useful in distinguishing which gene is affected in GA type II patients. Three sub-categories of GA type II are recognized, each of which is based on severity and presentation of clinical symptoms. (Frerman and Goodman 2014).

Patients in the first group present within the first 48 hours of life with severe hypoketotic hypoglycemia, hypotonia, metabolic acidosis, possibly hepatomegaly and a “sweaty feet” odor similar to that observed in isovaleric acidemia patients, and multiple congenital anomalies, such as dysplastic kidneys, facial dysmorphism, rocker bottom feet, abdominal wall defects and abnormal external genitalia. Such patients typically die within the first week of life (Olsen et al. 2003; Schiff et al. 2006; Frerman and Goodman 2014; Xi et al. 2014).

Patients in the second group present similarly to those in the first group, but without congenital anomalies. If these patients survive beyond the first week of life, they typically succumb within the first few months, often due to cardiomyopathy (Olsen et al. 2003; Schiff et al. 2006; Frerman and Goodman 2014; Xi et al. 2014).

The patients in the third group have a more mild form of GA type II, with onset anywhere from infancy to adulthood. Clinical presentation also varies widely in this group, and may include vomiting, hypoglycemia, metabolic acidosis, hepatomegaly, progressive lipid storage proximal myopathy and exercise intolerance. Symptoms in the third group often present in an episodic manner, making biochemical analysis challenging as abnormalities may be detectable only during periods of metabolic crisis (Olsen et al. 2003; Schiff et al. 2006; Frerman and Goodman 2014; Xi et al. 2014).

In all GA type II patients, generalized aminoacidemia and aminoaciduria, hyperammonemia and metabolic acidosis may be observed. Increased levels of sarcosine in both the serum and urine are common in those with mild, later onset GA type II. Biochemically, GA type II can be distinguished from GA type I based on the presence of 2-hydroxyglutaric acid (3-hydroxyglutaric acid is present in GA type I patients) (Frerman and Goodman 2014).

To date, no effective treatments are available for patients with the severe, infantile onset forms of GA type II. Some patients with the mild, later onset form have been shown to respond well to riboflavin, glycine and L-carnitine supplementation, as well as to dietary restriction of fat and protein (Olsen et al. 2007; Wen et al. 2013; Frerman and Goodman 2014). The majority of patients reported with the late-onset, riboflavin responsive form of GA type II have had defects in the ETFDH gene (Olsen et al. 2007; Wen et al. 2010; Cornelius et al. 2012; Wen et al. 2013).

Genetics

Glutaric acidemia type II is an autosomal recessive disorder caused by pathogenic variants in the ETFA, ETFB or ETFDH genes. To date, more than 10 causative variants have been reported in the ETFB gene (Human Gene Mutation Database). The majority of reported pathogenic variants are missense variants and small deletions, although nonsense and splice variants have also been reported. The variants are spread throughout the gene.

The ETFB gene is located on chromosome 19 and contains 6 exons. ETFB encodes the β-subunit of the electron transfer flavoprotein (ETF). The ETF protein is found in the mitochondrial matrix as a heterodimer, comprised of one subunit each of α- and β-monomers. ETF accepts electrons from at least 12 other flavoprotein dehydrogenases, then transfers them to the mitochondrial respiratory chain via the electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), encoded by the ETFDH gene. Clinical symptoms observed in ETFB deficient patients are due to a disruption of electron transfer to the respiratory chain (Cornelius et al. 2012; Frerman and Goodman 2014).

Testing Strategy

This test involves bidirectional Sanger sequencing using genomic DNA of all coding exons of the ETFB gene plus ~10 bp of flanking non-coding DNA on each side. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.

Indications for Test

Individuals with a positive newborn screening result for glutaric acidemia type II are good candidates for this test, as are individuals that exhibit biochemical and clinical symptoms of GA type II. Family members of patients known to have ETFB variants are also good candidates, and we will also sequence the ETFB gene to determine carrier status.

TEST METHODS

Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org). As required, DNA is extracted from the patient specimen. PCR is used to amplify the indicated exons plus additional flanking non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions. In nearly all cases, the full coding region of each exon as well as 10 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 10 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

Ship blood tubes at room temperature in an insulated container. Do not freeze blood.

During hot weather, include a frozen ice pack in the shipping container.
Place a paper towel or other thin material between the ice pack and the blood tube.

In cold weather, include an unfrozen ice pack in the shipping container as insulation.

At room temperature, blood specimen is stable for up to 48 hours.

If refrigerated, blood specimen is stable for up to one week.

Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.

For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.

DNA may be shipped at room temperature.

Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.

We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

PreventionGenetics should be notified in advance of arrival of a cell culture.

Culture and send at least two T25 flasks of confluent cells.

Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.

Send specimens in insulated, shatterproof container overnight.

Cell cultures may be shipped at room temperature or refrigerated.

Label the flasks with the patient name, date of birth, and/or ID number.

We strongly recommend maintaining a local back-up culture. We do not culture cells.